Competitive Effects of Anions on Protein Solvation by Aqueous Ionic Liquids

The present study utilizes molecular dynamics simulations to examine how different anions compete for protein solvation in aqueous solutions of ionic liquids (ILs). Ubiquitin is used as model protein and studied in IL mixtures sharing the same cation, 1-ethyl-3-methylimidazolium (EMIM), and two different anions in the same solution, from combinations of dicyanamide (DCA), chloride (Cl), nitrate (NO3), and tetrafluoroborate (BF4). Our findings reveal that specific interactions between anions and the protein are paramount in IL solvation, but that combinations of anions are not additive. For example, DCA exhibits a remarkable ability to form hydrogen bonds with the protein, resulting in a significantly stronger preferential binding to the protein than other anions. However, the combination of DCA with NO3, which also forms hydrogen bonds with the protein, results in a smaller preferential solvation of the protein than the combination of DCA with chloride ions, which are weaker binders. Thus, combining anions with varying affinities for the protein surface modulates the overall ion accumulation through nonadditive mechanisms, highlighting the importance of the understanding of competition for specific interaction sites, cooperative binding, bulk-solution affinity, and overall charge compensations, on the overall solvation capacity of the solution. Such knowledge may allow for the design of novel IL-based processes in biotechnology and material science, where fine-tuning protein solvation is crucial for optimizing performance and functionality.

showcases solvation parameters for ionic liquids (ILs) in six distinct concentrations, with A) 0.5, B) 1.0, C) 1.5, D) 2.0, E) 2.5, and F) 3.0 mol L-1 solutions, providing a comparative analysis of IL behavior across varying solute concentrations.verview of simulation boxes and component counts for initial system setups in all simulated systems.This table presents the dimensions of each simulation box, determined post-NPT equilibration, along with the number of components used to construct the initial systems.Table S3.Classification criteria based on Visual Molecular Dynamics (VMD) program.Basic residues are those positively charged due to their ability to accept protons.Acidic residues are characterized by a negative charge resulting from their propensity to donate proteins.Polar residues are those uncharged.Neutral residues are those nonpolar and characterized by their hydrophobic nature, not forming hydrogen bonds or interacting favorably with water.This classification is pivotal as it relies on the intrinsic side chain properties of each residue, particularly their capability to donate or accept protons, thereby dictating their interaction within the molecular environment.During system preparation, we employ the GROMACS tool, pdb2gmx, which automatically assigns protonation states to the residues.This assignment is based on canonical pKa values, with the system's pH assumed to be 7.
Aspartic acid (D) and Glutamic acid (E) fall into this category.
This category encompasses Serine (S), Threonine (T), Asparagine (N), and Glutamine (Q), among others.3.0 14.6 ± 0.9 13.3 ± 0.9 15.2 ± 0.4 13.9 ± 0.6 Table S6.DCA -water coordination numbers far from the protein.These are the number of water molecules within 10.0Å of the anion, considering the DCA molecules that have no atom within 30Å from the protein surface.RC is the reference IL concentration, and the anions present in each system are shown.The cation is EMIM in all systems.The data shown is the average and standard deviation of 20 replicas for each system.The integrals are calculated over lengths that ensure proper convergence for the majority of the systems.Subfigures contain the total KB integral for the anions in systems with A) 0.50, B) 1.00, C) 1.50, D) 2.00, E) 2.50 and F) 3.00 mol L -1 IL concentration.

Figure S1 .
Figure S1.Distribution functions of minimum distances in systems simulated with mixtures of ionic liquids.This figure shows the recalculated concentrations following system equilibration.The curves are displayed up to 10 Angstroms to enhance the visualization of key peaks.Subfigures A), B), and C) represent EMIMDCA + EMIMBF4; D), E), and F) correspond to EMIMDCA + EMIMNO3; and G), H), and I) EMIMBF4 + EMIMNO3.

Figure S2 .
Figure S2.Distribution functions of minimum distances in systems simulated with mixtures of ionic liquids.This figure shows the recalculated concentrations following system equilibration.To enhance visualization of key peaks, the curves are displayed up to 10 Angstroms.Subfigures A), B), and C) represent EMIMCl + EMIMDCA; D), E), and F) correspond to EMIMCl + EMIMNO3; and G), H), and I) EMIMCl + EMIMBF4.

Figure S3 .
Figure S3.Kirkwood-Buff integrals for all simulated systems with two types of anions.The integrals are calculated over distances that ensure proper convergence for the majority of the systems.Subfigures contain the total KB integral for the anions in systems with A) 0.50, B) 1.00, C) 1.50, D) 2.00, E) 2.50 and F) 3.00 mol L -1 IL concentration.The KB integrals for the cations or for the anion pairs converge to the same values, because of the necessary electroneutrality of the bulk solution in the presence of a solute with no net charge.

Figure S4 .
Figure S4.Distribution functions of minimum distances in systems simulated with one ionic liquid.This figure shows the recalculated concentrations following system equilibration.To enhance visualization of key peaks, the curves are displayed up to 10 Angstroms.Subfigures A), represent EMIMDCA; B) EMIMBF4; C) EMIMNO3; D) EMIMCl.

Figure S5 .
Figure S5.Kirkwood-Buff Integrals for all simulated systems with a single type of anion.The integrals are calculated over lengths that ensure proper convergence for the majority of the systems.Subfigures A) and B), represent EMIMDCA; C) and D), represent EMIMBF4; E) and F), represent EMIMNO3; G) and H) represent EMIMCl.

Figure S6 .
Figure S6.Density of chloride in the 2.0 mol L -1 solution containing DCA. Chloride is found to form electrostatic interactions with positively charged residues, as expected, but these interactions are much weaker than DCA hydrogen bonds.

Figure S7 .
Figure S7.Visualization of IL preferential solvation parameters in single-IL systems at different concentrations: showcases solvation parameters for ionic liquids (ILs) in six distinct concentrations, with A) 0.5, B) 1.0, C) 1.5, D) 2.0, E) 2.5, and F) 3.0 mol L -1 solutions, providing a comparative analysis of IL behavior across varying solute concentrations.

Figure S8 .
Figure S8.Protein root-mean-square deviation (RMSD) throughout the simulations.The average RMSD for 20 replicas is shown for each system.RMSD values consistently remain around 1.5 Ångströms for the protein backbone.This steady fluctuation within a confined range highlights the minimal structural changes the protein undergoes, ensuring its stability and retention within the native conformational ensemble.

Figure S9 .
Figure S9.example of the computation of the water coordination numbers of DCA, NO3 and EMIM, as a function of the distance to the protein, in the system with DCA and NO3 at 2.0 mol L -1 .The CN reported consists of the number of water molecules within 5Å of the ions, per ion.

Figure S10 .
Figure S10.Water Kirkwood-Buff integrals for all simulated systems with two types of anions.The integrals are calculated over lengths that ensure proper convergence for the majority of the systems.Subfigures contain the total KB integral for the anions in systems with A) 0.50, B) 1.00, C) 1.50, D) 2.00, E) 2.50 and F) 3.00 mol L -1 IL concentration.

Table S2 .
IL preferential solvation parameters in mixed-IL systems at different concentrations:

Table S4 .
Evaluation of Lennard-Jones and Coulomb interactions between proteins and ions, for species positioned with at least one atom from a protein atom closer than 10Å (molecules are not broken-apart in this computation by a strict cutoff).Calculations were conducted using the SolventShellInteractions.jl package, developed by our group, which processes output files from GROMACS.Reported energies include standard deviations across all replicas.The table categorizes anions into two types: anion 1 and anion 2, representing the respective anions from the first and second ionic liquids in each studied mixture.For clarity, within the mixture "EMIMDCA + EMIMCl," DCA is denoted as Anion 1, and Cl as Anion 2, illustrating the labeling convention used.

Table S5 .
DCA -water coordination numbers far from the protein.These are the number of water molecules within 5.0Å of the anion, considering the DCA molecules that have no atom within 20Å from the protein surface.RC is the reference IL concentration, and the anions present in each system are shown.The cation is EMIM in all systems.The data shown is the average and standard deviation of 20 replicas for each system.